VALIDATION OF AN IN-VITRO TERATOLOGY SYSTEM USING CHIRAL SUBSTANCES -STEREOSELECTIVE TERATOGENICITY OF 4-YN-VALPROIC ACID IN CULTURED MOUSE EMBRYOS

In vitro systems are important for toxicity testing as well as for inv estigating the mechanism of action of xenobiotics. The validation of s uch in vitro systems is often incomplete and extrapolation to the in v ivo situation is equivocal. In the present study, we studied the effec ts of enantiomers of an analogue of the antiepileptic drug valproic ac id (VPA): R(+)- and S(-)-4-yn-VPA (R- and S-2-n-propyl-4-pentynoic aci d), which have previously been shown to induce selective teratogenicit y in mice after in vivo administration, in mouse whole-embryo culture (WEC). Aqueous solutions of the sodium salts of the pure R-and S-enant iomers as well as R,S-4-yn-VPA (racemic mixture) or VPA itself were ad ded to the culture medium at 0, 0.075, 0.15, 0.3, 0.6, or 1.2 mmol/lit er and embryos were evaluated 24 hr later. The S-4-yn-VPA enantiomer i nduced clear concentration-dependent dysmorphogenesis that was evident even at the lowest concentration. The primary anomalies were neural t ube defects, erratic neural seams, blisters, and rotational defects. E mbryolethality was observed at 1.2 mmol/liter. The R-4-yn-VPA enantiom er was neither embryotoxic nor dysmorphogenic at any tested concentrat ion. The lack of biological activity over 24 hr in WEC with the R-enan tiomer suggests also that, as previously shown in vivo, there was no r acemization of this isomer to the more active S-enantiomer. The racemi c mixture of R and S isomers appeared to be slightly more embryolethal and dysmorphogenic than VPA. Overall, the potency of the S-enantiomer was approximately four times that of VPA. Therefore, the rank order o f the four chemicals tested was S(-) much greater than S(-), R(+) > VP A much greater than R(+), which is in agreement with the effects obser ved in in vivo exposed mice. These data demonstrate a direct stereosel ective effect of these compounds on the embryo. This is the first illu stration of the stereoselectivity of a xenobiotic in the WEC in vitro test system. Pure and stable enantiomers, which induce stereoselective toxicity in vivo, are demonstrated to be valuable for validation of t his in vitro system. (C) 1995 Academic Press, Inc.